In this resubmission of our joint PI R21 grant application, we propose to investigate the role of a newly-discovered type II procollagen mRNA transcripts, termed IIC, with respect to skeletal development. Previous work in other labs have shown that the type II procollagen gene (Col2a1) can be alternatively spliced to produce two isoforms by inclusion (type IIA) or exclusion (type IIB) of exon 2, which encodes a cysteine-rich domain within the amino propeptide of the procollagen molecule. In addition, this is a developmentally-regulated event where chondroprogenitor cells synthesize mainly type IIA mRNA, while differentiated chondrocytes produce mainly type IIB mRNA. We recently discovered two additional Col2a1 isoforms during in vitro chondrogenesis assays that we have named IIC and IID. IIC mRNA contains only the first 34 nucleotides of exon 2 by use of an alternative splice site in exon 2 resulting in a premature termination codon. IID mRNA contains an extra three nucleotides at the end of exon 2 that encodes either tryptophan (in human) or arginine (in mouse) without altering the subsequent protein reading frame. We hypothesize that the IIC splicing event is an essential mechanism required for proper formation of cartilage (and possibly other tissues) during embryonic development. We therefore propose to (1) generate a recombinant knock-in mouse model with an inactivated IIC splice site, and (2) To study the regulation and function of Col2a1 IIC mRNA during chondrogenesis in vitro. This is an elegant approach, as in vivo and in vitro we will specifically inhibit production of one mRNA isoform while retaining the cell's ability produce the other three Col2a1 isoforms. Our preliminary in vitro studies using a mini-gene construct has shown that deleting the IIC splice site results in a significant change in the ratio of IIA/IID to IIB. The function of the untranslated IIC isoform may be to regulate the proportions of the other translated alternatively spliced Col2a1 isoforms. Therefore, we can predict the effect of IIC inhibition upon production of other Col2a1 isoforms, and on cartilage development in the knock-in mouse model. We believe that the IIC transcript functions only at the level of mRNA and is rapidly degraded soon after it is synthesized. We plan to investigate whether IIC mRNA abundance is regulated by a process called nonsense-mediated decay (NMD), which occurs iwith other mRNAs that have similar regulatory roles. Using a cell line that undergoes chondrogenic differentiation when treated with insulin (ATDC5) we will transfect these cells with antisense oligonucleotides that will block splicing at the IIC splice site. This should have the same effect upon chondrogenesis as in the knock-in mouse, which has a mutated IIC splice site. This in vitro system will allow us to study the effect of preventing IIC mRNA production upon production of the other Col2a1 soforms. The innovative methods of manipulating a splice site in order to generate a knock-in mouse, and blocking a splice site in vitro, will prove to be a useful strategy to study any gene that is subject to alternative splicing. PUBLIC HEALTH RELEVANCE: This grant proposal will investigate the significance of novel type II collagen (Col2a1 gene) alternative transcripts within the context of skeletal development, primarily by generation of a recombinant mouse model. Very few mouse models to study the importance of alternative splicing sites have been developed to date. Since alternative splicing of collagen type II mRNA is a key element of skeletogenesis, the development of a knock-in model to study the role of collagen II splicing in skeletal development is an important step forward in the field of connective tissue biology. The importance of alternative splicing in the production of genetic diversity has become evident in recent years, and alternative splicing-related knock-in mice will be required for numerous other genes in the future. We also anticipate that the mouse model developed in this study will shed light upon other developmental processes that have been shown to involve expression of type II collagen. Type IIA procollagen mRNA is expressed at relatively low levels during development in a number of embryonic non-cartilage tissues such as the notochord, somitic mesoderm, fetal brain, spinal cord, developing eye, tendons and ligaments. Additional work on this project will investigate the mechanisms for regulating the amount of each alternatively spliced mRNA type during differentiation and development.